1. Field of the Invention
The present invention relates generally to compositions and methods for the treatment and diagnosis of lipopolysaccharide (LPS)-associated diseases, such as Gram-negative sepsis. More particularly, the present invention relates to the preparation and use of certain mammalian cationic proteins for such treatment and diagnosis.
A rational approach for the control of Gram-negative sepsis is to neutralize the toxic effects of lipopolysaccharides which are released during the treatment of the underlying bacterial infection. Cationic antibiotics, such as polymyxin B, bind to and neutralize some types of lipopolysaccharide, but their use is limited by toxicity. Certain LPS-neutralizing monoclonal antibodies recognize common types of LPS, but are not effective against all species of Gram-negative bacteria. Antibodies made to regions of lipopolysaccharide conserved among many species, such as the lipid A core, appear to be ineffective against certain types of LPS. Certain mammalian polypeptides have been identified that bind to LPS. These include LPS-binding protein (LBP) and bactericidal permeability increasing protein (BPI). BPI can neutralize the toxic effects of LPS.
Of particular interest to the present invention, a cationic protein having a molecular weight of approximately 16.6 kD has been identified in rabbit granulocytes. The protein, known as CAP18, has been found to bind to and attenuate the activity of LPS in certain in vitro assays.
For purposes of human therapy and diagnosis, however, it is frequently preferable to utilize a human form of a protein rather than a mammalian homolog. This is particularly true when the human form of the protein differs significantly from other mammalian forms of the homologous proteins. Identification of the human form of a protein, however, can be difficult even when a protein has been isolated and cloned in other mammals, particularly when the amino acid sequence of the human homolog differs significantly from the mammalian prototype(s). It is often desirable to provide human or humanized proteins which are improved in some way over the natural form of the protein. For example, proteins and polypeptides useful for therapy and diagnosis may be truncated or shortened forms of the natural human or other mammalian protein. The therapeutic forms of the proteins and polypeptides may also be modified at certain residues in order to enhance activity of the protein in some desirable manner.
It would be desirable to provide improved methods for the treatment of lipopolysaccharide-associated diseases, such as Gram-negative sepsis. In particular, it would be desirable to identify compositions and methods capable of inhibiting or neutralizing lipopolysaccharide-associated damage which occurs in such diseases. It would further be desirable to provide an isolated and purified mammalian cationic protein capable of binding to lipopolysaccharide and inhibiting or neutralizing its activity. It would be particularly desirable to provide a synthetically or recombinantly produced mammalian cationic protein, such as mammalian CAP18. It would be further desirable to provide isolated and purified human cationic proteins capable of binding to lipopolysaccharide and inhibiting or neutralizing its activity. It would be still further desirable to provide modified forms of human and other mammalian cationic proteins which possess lipopolysaccharide binding activity in combination with other desirable activities, such as anti-coagulation activity.
2. Description of the Background Art
Hirata et al., AGGLUTINATION OF ERYTHROCYTES SENSITIZED WITH RE-LPS ELICITED BY CATIONIC PROTEIN FROM RABBIT GRANULOCYTES, in Endotoxin: Structural Aspects and Immunobiology of Host Responses; Meeting Abstract Riva del Sole, Giovinazzo (BARI), Italy, May 29-June 1, 1986, describes the extraction of cationic proteins from rabbit peritoneal granulocytes capable of agglutinating red blood cells sensitized with lipopolysaccharide. Tsunoda et al. (1987), Abstract, International Conference on Endotoxins II, Amsterdam, the Netherlands, May 22-23; page 79, describes activities of a cationic protein from rabbit granulocytes, including anticoagulant activity. Yoshida et al. (1988) Abstract, International Symposium on Endotoxin, Osaka, Japan, May 16-19, page SII-3, describes granulocyte cationic proteins having molecular weights from 2 kD to 70 kD. Shimomura et al. (1989), Abstract 1228, XIIth Congress of the International Society on Thrombosis and Haemostasis, Tokyo, Japan, August 19-25, page 339, describes the anticoagulant and antibacterial activities of rabbit granulocyte cationic proteins. Hirata et al. (1990), Abstract S33-3, IUMS Congress: Bacteriology & Mycology, Osaka, Japan, September 16-22, page 19, describes a cationic protein from rabbit granulocytes having an estimated molecular weight of 18 kD. Hirata et al. (1990), Abstract II-P-87, The First Congress of the International Endotoxin Society, San Diego, Calif., May 10-12, also describes the 18 kD cationic protein from rabbit granulocytes.
Hirata et al. (1990), Endotoxin, Friedman et al., eds., Plenum Publishing Co., pages 287-299, describes the partial purification of cationic protein from rabbit granulocytes (rabbit CAP18). The cloning and expression of rabbit CAP18 are described in Larrick et al. (1991) Biochem. Biophys. Res. Comm. 179:170-175. The cloning and expression of mammalian CAP18 are described in co-pending applications Ser. No. 07/916,761 and Ser. No. 07/916,765, the full disclosures of which are incorporated herein by reference. These applications also describe an active carboxy-terminal fragment of CAP18 designated the reactive nitrogen inhibitory protein (RNIP). Saba et al. (1967) J. Clin. Invest. 46:580-589 and (1968) Blood 31:369-380, described the presence of cationic proteins having anti-coagulant activity in granulocytes.